NIS2+™, an effective blood-based test for the diagnosis of at-risk nonalcoholic steatohepatitis in adults 65 years and older

Background: Older patients are at increased risk for at-risk NASH, defined as NASH with NAFLD activity scores (NAS) ≥4 and significant fibrosis (F ≥ 2). The aim of this study was to compare the performance of 2 new blood tests, NIS4® and NIS2+™, with FIB-4, NFS, ELF™, and alanine aminotransferase (ALT) for the diagnosis of at-risk NASH in a cohort of patients aged ≥65 years. Methods: The clinical performance of multiple blood-based tests was assessed for their ability to detect at-risk NASH using the RESOLVE-IT diag cohort, a large population of patients with metabolic risk who were screened for potential inclusion in the RESOLVE-IT phase 3 trial. Results: The study cohort (n = 2053) included patients with the full histological spectrum of NAFLD, with patients having liver fibrosis stages F0–4 and NAS scores 0–8. NIS4® and NIS2+™ showed similar assay performance in patients who were <65 versus ≥65 years of age (AUROC = 0.80 vs. 0.78, p = 0.47; 0.81 vs. 0.83 p = 0.45, respectively) for the identification of at-risk NASH. In patients ≥65 (n = 410), NIS2+™ exhibited the highest AUROC compared to NIS4®, FIB-4, NFS, ELF™, and ALT (AUROC = 0.83 vs. 0.78, 0.68, 0.58, 0.69, 0.74, respectively; all p ≤ 0.0009). For NIS2+™, the sensitivity and NPV for ruling-out at-risk NASH at the 0.46 cutoff were 90.2% and 86.0%, and the specificity and PPV for ruling-in at-risk NASH at the 0.68 cutoff were81.1% and 76.3%, respectively. Conclusions: The clinical performance of NIS2+™ was superior for the diagnosis of at-risk NASH in patients ≥65 years of age. These data support the clinical value of this blood-based test for the diagnosis of at-risk NASH in older adults.


INTRODUCTION
NAFLD is becoming the leading cause of chronic liver disease worldwide. [1][2][3] In the United States alone, the prevalence of NAFLD and NASH has increased over the past decade, culminating in an estimated prevalence of 25%-30% and 2.5%-5%, respectively. [1,[4][5][6][7][8] The most common diseases associated with NAFLD and NASH are cardiometabolic diseases such as type 2 diabetes (T2D), obesity, hypertension, and dyslipidemia, which are also growing in prevalence in the United States and globally. [2,9] It has been estimated that up to 20% of adults with NAFLD have NASH, and NASH has become the leading cause of chronic liver disease. [10,11] At-risk NASH, which is defined as having NASH with NAFLD activity scores (NAS) ≥ 4 and significant liver fibrosis (F ≥ 2), based on histological scoring of liver biopsy, is associated with increased risk of disease progression as well as liver-related and all-cause mortality. [12][13][14][15] Recently, the prevalence of at-risk NASH was shown to be about 4.4% in the general United States population and up to 18.3% in patients with T2D. [7] Along with the increased prevalence in the United States, NASH has become the second leading cause of end-stage liver disease and the fastest-growing etiology of HCC and need for a liver transplant. [1,[16][17][18][19][20] Older adults, in particular those on Medicare, have an even higher risk for NAFLD-mediated and NASH-mediated progression to cirrhosis, acute on chronic liver failure, liver transplantation, and HCC. [21][22][23][24] A recent article reported that NAFLD is the leading etiology of HCC among Medicare recipients (2011)(2012)(2013)(2014)(2015), accounting for 35.6% of the HCC cases. [24] In turn, among Medicare recipients with HCC, NAFLD was the most common cause of mortality. [25] Compared to other etiologies, NAFLD was associated with lower surveillance for HCC, reduced early detection, and poorer overall survival. [21,24] In addition, NASH exceeded all other causes of liver transplantation in Medicare patients in 2019 and, along with alcohol-associated hepatitis, is considered one of the most rapidly increasing etiologies of liver transplantation. [23,26] The proportion of patients with NASH among elderly liver transplant candidates increased from 13% in [2002][2003][2004][2005] to 39% in 2018-2020. [23] Moreover, a recent study in Medicare recipients (2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015) showed that older patients were likely to be diagnosed with cirrhosis before being diagnosed with NASH and that later-stage diagnosis of patients with NASH led to a substantial increase in health care resource utilization and costs. [27] Therefore, it is critical to identify older adults with NASH in order to treat and reduce the progression to cirrhosis, HCC, and the need for a liver transplant.
NIS4 ® is a blood test that combines the results of 4 biomarkers (miR-34a-5p, YKL-40, α2-macroglobulin, and glycated hemoglobin [HbA1c]) into a single score that aids in the diagnosis of at-risk NASH. [28] In the noninvasive metabolic liver disease assessment study, which was conducted by the Foundation for the National Institutes of Health Biomarkers Consortium and the NASH Clinical Research Network, NIS4 ® achieved significantly higher performance than alanine aminotransferase (ALT) and OWLiver for the diagnosis of patients with NASH. [29,30] For the diagnosis of significant fibrosis (F ≥ 2), NIS4 ® outperformed Fibrosis-4 (FIB-4), Enhanced Liver Fibrosis test (ELF™), and Pro-Peptide of Type III Collagen (Pro-C3), albeit the latter tests were originally designed for the detection of advanced liver fibrosis (stage F ≥ 3). [30] Importantly, NIS4 ® was significantly better than ALT and FIB-4 for the identification of at-risk NASH. [29,30] Recently, NIS2+™ was developed as an optimization of the NIS4 ® technology by removing α2-macroglobulin and HbA1c from the equation and combining the results for miR-34a-5p and YKL-40 with a sex correction for miR-34a-5p, in order to robustly detect at-risk NASH, irrespective of patient characteristics such as age, sex, BMI, or T2D status. [31] Previously published data showed that NIS2+™ had a high overall clinical performance for the detection of at-risk NASH, achieving an area under the receiver operating characteristic curve (AUROC) of 0.81 in a large study population. [31] For the purposes of reimbursement, the Centers for Medicare & Medicaid Services in the United States requires peer-reviewed, published data showing that tests that will be used for patient management have good assay performance in patients who are ≥ 65 years of age. To date, the robustness of the NIS4 ® and NIS2+™ tests has not been assessed specifically in patients who are ≥ 65 years of age. Therefore, the first aim of this study was to perform a retrospective, crosssectional analysis assessing the performance of NIS4 ® and NIS2+™ in patients <65 and ≥ 65 years of age in order to ensure the 2 tests detect at-risk NASH equivalently in both younger and older individuals. The second aim was to compare NIS4 ® , NIS2+™, FIB-4, NAFLD fibrosis score (NFS), ELF™, and ALT in a cohort of patients ≥ 65 years of age in order to determine which test had the highest overall performance for detecting at-risk NASH in elderly patients who would benefit from intensive lifestyle or therapeutic interventions.

Study population
The RESOLVE-IT diag cohort consisted of patients screened for, and who may or may not have eventually been enrolled in, the RESOLVE-IT trial (NCT02704403), a multicenter, randomized, double-blind, placebo-controlled phase 3 trial designed to evaluate the efficacy and safety of elafibranor in patients with NASH and liver fibrosis (n > 5000 patients). [28] Including patients who were screened for, but may not have been included in, the RESOLVE-IT trial enabled the current study to include subjects that were balanced across the histological endpoints of NASH and liver fibrosis. It also empowered the study to include a larger number of patients ≥ 65 years of age compared to the RESOLVE-IT trial alone. Patients with available biopsy results and with full data for NIS4 ® (Genfit SA), NIS2+™ (Genfit SA), FIB-4, NFS, ALT, and ELF™ (Siemens Healthineers) at baseline were included in this analysis. To ensure the relevance of the biomarker concentrations versus histology, only patients with a gap of ≤ 90 days between biopsy and blood sample dates were retained. This process of selection led to a study cohort of n = 2053 patients, which was then divided into 2 groups: <65 (n = 1643) and ≥ 65 (n = 410) years of age. Patients in the study cohort were recruited at 249 global centers (including the United States and Europe) and screened between June 2016 and March 2020. The percentage of patients from the United States in the total study cohort was 46% (947/2053) and in those who were ≥ 65 years of age was 50% (205/410), the latter representing the Medicare population. The inclusion criteria for initial screening of the RESOLVE-IT diag cohort included patients 18-75 years of age with one or more metabolic disease risk factors (eg, T2D, obesity, dyslipidemia, and hypertension). Patients were excluded if daily alcohol consumption was greater than 2 drink units per day (20 g) in women and 3 drink units per day (30 g) in men, hepatosteatosis was due to secondary causes, or they had other chronic liver diseases. During the screening stage, clinical data, blood samples, and liver biopsy results were collected for all patients included in this study. Liver biopsies were scored based on the NASH Clinical Research Network classification system. Histological scoring was performed by a single trained pathologist at Hôpital Beaujon (Paris, France), who was blinded to the clinical and biochemistry data. All research for this study was conducted in accordance with both the Declarations of Helsinki and Istanbul. The clinical study protocol was approved in all countries by national authorities and institutional ethics review committees, and all patients provided written informed consent for participation in the trial and collection of blood samples for biomarker research. The names of the IRB committees and the approval numbers are available upon request.

Clinical characteristics and laboratory tests
Demographic, medical history, and anthropometric characteristics, including body weight, height, age, sex, and BMI, were collected at clinical sites from each patients' medical record. Blood samples were collected after fasting for at least 12 hour. Biochemical analyses were performed using blood samples collected at a median time and mean time of 25 and 16 days, respectively, after biopsy. Hematology and biochemical analyses, including α2-macroglobulin and HbA1c, were conducted by BARC-Europe (Ghent, Belgium). All biochemical analyses were performed using commercially available kits or reagents. YKL-40 (DC3L10 kit; Bio-Techne, Minneapolis, MN) and miR-34a-5p assays were performed in the laboratory at Genfit SA (Loos, France). Details of the miR-34a-5p assay and the development of the NIS4 ® and NIS2+™ algorithms, including the determinations of the cutoffs, were reported. [28,31] Neither vibration-controlled transient elastography (VCTE) Fibro-Scan nor magnetic resonance elastography results were available for many of the participants; therefore, VCTE results were not used for comparison purposes. Patients with at-risk NASH were defined as those having NASH with NAS ≥ 4 and liver fibrosis stage F ≥ 2.

Statistical analysis
Descriptive statistics were generated for baseline characteristics of the full cohort, and patients aged <65 and    , and positive predictive value (PPV) for ruling-in and ruling-out at-risk NASH. [28,[31][32][33]35] Upper limit normal values for ALT of 33 IU/L for women and 41 IU/L for men have been used as single cutoff values for ruling-in and ruling-out atrisk NASH. The Youden Index statistic, another measure of a test's effectiveness, was defined as the sum of specificity and sensitivity minus 1, which could be used to derive an optimal cutoff for a biomarker, also called Youden cutoff, in that it would maximize the Youden Index statistic. The Youden Index cutoffs were determined using the full study cohort (n = 2053) for the detection of at-risk NASH in order to provide a more reliable comparison of sensitivity, specificity, NPV, and PPV values among the tests for the same endpoint instead of those published for the detection of advanced fibrosis regarding FIB-4, NFS, and ELF™.
Sensitivity and specificity were compared using McNemar tests, and PPV and NPV were compared using Generalized Score tests (

Clinical characteristics of the study population
A comparison of the subpopulation of study participants who were ≥ 65 versus those who were <65 years of age based on baseline demographics, clinical characteristics, and laboratory results is provided in Table 1. Patients who were ≥ 65 were more likely to have T2D, dyslipidemia, arterial hypertension, lower BMI, and obesity compared to those who were < 65 years. They were also more likely to have a higher aspartate aminotransferase (AST)/ALT ratio, fasting plasma glucose (FPG), HbA1c, and lower ALT, total cholesterol, and platelet count. While the median model for end-stage liver disease (MELD) score was similar, more patients who were ≥ 65 were in the higher MELD score classes. While the percentages of adults with NAFLD, NASH, and at-risk NASH were similar between adults who were <65 versus ≥ 65 years, patients who were ≥ 65 years had a higher mean liver fibrosis stage compared to those who were <65 years (2.16 vs. 1.79, p < 0.0001) and had a lower mean NAS (3.84 vs. 4.17, p = 0026) based on liver biopsy results (Table 1).
Variables that were non-normally distributed were log-transformed and are reported as median (IQR Within the ≥ 65 age subpopulation (n = 410), the prevalence of at-risk NASH was 47% (193/410), with a lower percentage of males in this group compared to those without at-risk NASH (50% vs. 73%, p < 0.0001) ( Table 2). A higher percentage of the at-risk NASH group were white (67% vs. 59%, p = < 0.0001) and had T2D (59% vs. 44%, p = 0.0039) as well as higher measures of AST, ALT, γ-glutamyl transferase (GGT), FPG, HbA1c, triglycerides, and total cholesterol compared to the non-at-risk NASH population. There was no statistically significant difference in the median MELD score between older patients with at-risk NASH and those with NASH, although those with at-risk NASH tended to be in a higher MELD score class. In addition, among older at-risk NASH patients, 58% had liver fibrosis stage F3 and 11% were cirrhotic (F4) (  (Table 3). For ruling-out at-risk NASH, the sensitivity and NPV for NIS4 ® at the low cutoff point of 0.36 was higher in patients aged ≥ 65 compared to those in patients < 65 years old (95.3% and 89.8% vs. 84.0% and 80.9%, respectively). While comparable results on sensitivity and NPV were obtained for NIS2+™ at the low cutoff point of 0.46 in between these 2 age groups, it was noticed that the difference in performance between the subpopulations was reduced compared to those associated with NIS4 ® . While both NIS4 ® and NIS2+™ exhibited a high specificity at the cutoff point for ruling-out at-risk NASH in patients <65 years of age (57.0% vs. 62.9%, respectively), NIS2+™ retained high specificity in the older age group while NIS4 ® was associated with a decreased specificity (53.9% vs. 36.4%, respectively) ( Table 3). For ruling-in at-risk NASH, the specificity and PPV for NIS4 ® at the high cutoff point of 0.63 were lower in patients aged ≥ 65 compared with those in patients < 65 years old, while NIS2+™ achieved similar performance among both subgroups at the high cutoff point of 0.68. Both NIS4 ® and NIS2+™ achieved good sensitivity for ruling-in atrisk NASH, in both age groups, with the performance ranging between 55.9% and 73.6%. However, while NIS2+™ exhibited similar sensitivities in both age groups (61.2% vs. 68.4%), NIS4 ® exhibited a notable difference across the 2 subgroups (55.9% vs. 73.6%) ( Table 3). Overall, while both tests achieved high performance in both age groups, NIS2+™ returned more robust/stable performance across age groups compared with NIS4 ® .
Similar data were computed for the identification of at-risk NASH with F ≥ 3 and F4 using NIS2+™ (Supplemental Table S1, http://links.lww.com/HC9/ A427). Overall, NIS2+™ kept stable clinical performance across age groups when identifying these endpoints, and was notably associated with an AUROC of 0.79 for the identification of at-risk NASH F ≥ 3 in patients aged ≥ 65 years of age. The low prevalence of at-risk NASH patients with cirrhosis in both age groups (4.4% and 5.1%) induced the low PPV and high NPV achieved by NIS2+™, and limited the interpretation of clinical performance due to the low number of cases in each age group (n = 72 and 21). However, it was interesting to note that no more than 4% to 5% of these at-risk NASH patients with cirrhosis were wrongly ruled out by NIS2+™ while more than 76% were accurately ruled in for both age groups.

Ability of NIS4 ® and NIS2+™ to differentiate between NAS scores and liver fibrosis stages
In the subpopulation of patients ≥ 65 years of age, the NIS4 ® and NIS2+™ mean scores were plotted by category of NAS (0-1, 2-3, 4-5, 6-8) and liver fibrosis stage (F0-F4) (Figure 2). There were significant differences in the NIS4 ® and NIS2+™ scores between the 4 categories of NAS, highlighting the ability of these tests to stratify patients by the metaboinflammatory component of their liver disease (Figure 2A, B). There were also differences in the NIS4 ® and NIS2+™ scores across the 4 stages of liver fibrosis, with both tests exhibiting the highest discrimination between stages 1 and 2 ( Figure 2C, D).

DISCUSSION
The current study is the first to show the clinical performance of NIS4 ® , and the improved performance of NIS2+™ compared to NIS4 ® , in a population of older adults ( ≥ 65 years of age) with at least 1 metabolic risk factor such as T2D, obesity, dyslipidemia, or hypertension. Based on their AUROC values, both NIS4 ® and NIS2+™ were able to identify patients with at-risk NASH equally well in patients who were <65 versus those who were ≥ 65 years. Furthermore, while both NIS4 ® and NIS2+™ may enable physicians to rule out or rule in at-risk NASH in a well-balanced manner with respect to sensitivity and specificity, NIS2+™ was associated with more robust and stable clinical performance across both age subpopulations. This is important because other blood-based tests for identifying patients with liver fibrosis, such as NFS and FIB-4, have shown differing diagnostic discrimination in older ( ≥ 65 y) versus younger (< 35 y) adults. [28,36,37] It has been suggested that for FIB-4, age-dependent cutoffs might be more appropriate. [36,37] However, age-dependent cutoffs for FIB-4 have not been standardized and were not recommended in the recent AASLD or American Association for Clinical Endocrinology guidelines; therefore, we used the more commonly used FIB-4 cutoffs for this study. [13,38] Both tests (NIS4 ® and NIS2+™) had much smaller indeterminate or moderate risk zones relative to other blood tests that were developed for advanced liver fibrosis. The data in this study confirm previously published assay performance data in independent patient cohorts. [28][29][30] Of all the blood-based tests that were previously compared, NIS2+™ exhibited the best clinical performance, with the highest AUROC and a more balanced sensitivity and specificity at the low, high, and Youden cutoff points. [31] The current study extends these observations by showing that NIS2+™ exhibits effective overall performance in a patient population that reflects the Medicare population in the United States. One of the strengths of NIS2+™ is the ability to identify at-risk NASH at a stage where lifestyle modification and/or therapeutic agents can reduce liver inflammation and fibrosis. Noninvasive tests, such as FIB-4 and ELF™, were designed to identify patients with advanced liver fibrosis and a high likelihood of progression to end-stage liver disease. Patients identified using these tests could be treated with drugs designed for reducing advanced liver fibrosis, which is a later disease stage. NIS2+™ test results, on the other hand, may permit physicians to more confidently identify patients earlier in the course of metabolic liver disease and consider prescribing intensive lifestyle or a therapeutic intervention specifically designed to treat the metaboloinflammatory component of NASH in order to reduce progression to end-stage liver disease.
Optimization of the NIS4 ® technology to produce the NIS2+™ test provided several advantages. As previously published, after optimization of the equation, HbA1c and α2-macroglobulin no longer contributed significantly to the prediction of at-risk NASH; therefore, they were removed from the equation. [31] Removing HbA1c also avoids the perceived issue that use of antidiabetic medications that reduce HbA1c levels may affect scores and may complicate the interpretation of the test result, especially in patients with uncontrolled HbA1c who require aggressive treatment. The further addition of a sex correction for miR-34a-5p enabled the NIS2+™ results to be independent of sex. Therefore, the NIS2+™ is more balanced than NIS4 ® with respect to age, sex, and T2D status; however, it retains the strong assay performance for the detection of at-risk NASH. Moreover, the ROC curve ( Figure 1) and clinical performance (Table 4) show that NIS2+™ has higher specificity for ruling in at-risk NASH than NIS4 ® ; however, it retains high sensitivity for ruling out at-risk NASH. In addition, NIS2+™ appears to better differentiate liver fibrosis stages F2-F4 from stages F0-F1 than NIS4 ® , speaking to the enhanced ability to differentiate patients with at-risk NASH from those with no to mild fibrosis ( Figure 2). Current AASLD and American Association for Clinical Endocrinology guidelines support screening of patients with multiple metabolic risk factors and/or T2D for NASH. [13,38] For the first time, the American Heart Association acknowledged in a published statement that NAFLD is a risk factor for atherosclerotic cardiovascular disease and that CVD is the principal cause of death in patients with NAFLD. [39] Several recent publications have also been directed toward primary care physicians and endocrinologists in order to increase awareness of the disease as well as provide guidance on how to evaluate and treat patients identified as having NAFLD/NASH. [40][41][42] Increased awareness of NAFLD/NASH and adherence to newly published guidelines will likely lead to a reduction in patient progression to cirrhosis, HCC, and liver transplantation. Moreover, the addition of new tests for the identification of patients with at-risk NASH, such as NIS2+™, should facilitate this goal by identifying patients in the primary care or endocrinology setting. By identifying patients earlier in their disease, there may also be a concomitant reduction in health care utilization and costs in a disease that is growing in prevalence yet remains somewhat silent, meaning that it is currently hard to detect until later in a patient's disease path.
The strengths of this study include the large number of patients who were ≥ 65 years of age as well as the fact that the study cohort was balanced with respect to the various stages of disease activity and liver fibrosis. In addition, patients were included with or without increased ALT or AST and came from multiple general and specialty practice settings including hepatology, gastroenterology, and internal medicine. These points are especially important because they substantiate the fact that NIS2+™ is able to identify patients with at-risk NASH among individuals with various stages of NAFLD, and could be used as a screening tool in a population of patients with metabolic risk factors.
The limitations to this study include the fact that the RESOLVE-IT diag cohort did not have data for Pro-C3, a biomarker of fibrosis that has been used in other NASH trials, nor did it have sufficient VCTE or magnetic resonance elastography data to compare with the results from the blood-based tests reported here. Because this data set did not have VCTE results for the majority of the participants, and no CAP scores, it was not possible to compare the blood-based tests with combined imaging and blood-based algorithms such as the FAST score.

CONCLUSIONS
The clinical performance of NIS2+™ was superior to that of NIS4 ® , FIB-4, NFS, ALT, and ELF™ for the identification of at-risk NASH in patients aged ≥ 65 years. Therefore, these data support the value of this test for the detection of at-risk NASH in older adults.

AUTHOR CONTRIBUTIONS
Jérémy Magnanensi contributed to the study design, data generation, data analysis, and publication writing. Margery A. Connelly contributed to the study design, data analysis, and publication writing. Arun J. Sanyal, Zouher Majd, Christian Rosenquist, Delphis M. Vera, and James P. Almas contributed to the data analysis and publication writing. All authors reviewed, edited, and approved the final version of the manuscript.